関 直彦

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein-coding genes. To identify miRNAs that have a tumor suppressive function in bladder cancer (BC), 156 miRNAs were screened in 14 BCs, 5 normal bladder epithelium (NBE) samples and 3 BC cell lines. We identified a subset of 7 miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b and miR-1990) that were significantly downregulated in BCs. To confirm these results, 104 BCs and 31 NBEs were subjected to real-time RT-PCR-based experiments, and the expression levels of each miRNA were significantly downregulated in BCs (p < 0.0001 in all). Receiver-operating characteristic curve analysis revealed that the expression levels of these miRNAs had good sensitivity (>70%) and specificity (>75%) to distinguish BC from NBE. Our target search algorithm and gene-expression profiling in BCs (Kawakami et al., Oncol Rep 2006;16:521-31) revealed that Keratin7 (KRT7) mRNA was a common target of the downregulated miRNAs, and the mRNA expression levels of KRT7 were significantly higher in BCs than in NBEs (p = 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p < 0.0001 in all). Gain-of-function analysis revealed that KRT7 mRNA was significantly reduced by transfection of 3 miRNAs (miR-30-3p, miR-133a and miR-199a*) in the BC cell line (KK47). In addition, significant decreases in cell growth were observed after transfection of 3 miRNAs and si-KRT7 in KK47, suggesting that miR-30-3p, miR-133a and miR-199a* may have a tumor suppressive function through the mechanism underlying transcriptional repression of KRT7. (C) 2009 UICC

In our microarray screening of methylated genes in bladder cancer (BC), the collagen type I alpha 2 (COL1A2) gene was the most up-regulated among the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC. We subjected a bladder cancer cell line (BOY) and 67 BC specimens and 10 normal bladder epitheliums (NBEs) to conventional or real-time methylation quantitative polymerase chain reaction (PCR) and to real-time reverse transcriptase (RT)-PCR. We also established a stable COL1A2 transfectant for evaluating cell proliferation and migration activity. After 5-aza-dC treatment, the expression levels of COL1A2 mRNA transcript markedly increased in BOY. Our cell proliferation assays consistently demonstrated growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (p<0.0001). Wound healing assays also showed significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (p=0.0016). We demonstrated by bisulfite DNA sequencing that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index of COL1A2 was significantly higher in the 67 BCs than in the 10 NBEs (p=0.0011). Conversely, COL1A2 mRNA transcript was significantly lower in the BCs than in the NBEs (p=0.0052). The mechanism of COL1A2 down-regulation in BC is through CpG hypermethylation of the promoter region. COL1A2 gene inactivation through CpG hypermethylation may contribute to proliferation and migration activity of BC.

While lymph node metastasis is a major factor associated with poor prognosis in cancer, little is known of its molecular mechanisms. The aim of this Study was to identify genes differentially expressed between non-cancerous and cancerous lung tissues, and to investigate the gene expression profiles of 41 primary lung adenocarcinomas to select sets of gene predictors for lymph node metastasis Of lung cancer. Gene expression Profiles were obtained using oligonucleotide microarrays, and predictor sets constructed by evaluating the statistical significance of expression levels of selected genes. Gene analysis revealed 15 predictor genes for lymph node metastasis of lung adenocarcinoma. Using the most suitable set of genes, it was possible to predict the lymph node metastasis of patients with lung cancer. The prediction scoring system yielded 71.4% accuracy for forecasting lymph node metastasis in 14 independent test cases. Survival was also significantly better in 18 cases that were pathologically LN negative and predicted to be LN negative according to molecular classification, compared with 23 cases that were pathologically LN positive or predicted to be LN positive according to molecular classification. Gene expression analysis combined with statistical analysis successfully distinguished lymph node metastasis. The findings of this study showed that pathological diagnosis combined with molecular classification clearly distinguished patients with good prognoses from patients with poor prognoses. (C) 2008 Published by Elsevier Ireland Ltd.

To identify putative biomarkers in squamous cell carcinoma (SCC), a survey of parallel chromosomal alterations and gene expression studies in 10 SCC cell lines were performed using array-comparative genomic hybridization (CGH) and oligomicroarray techniques. The most frequent changes were gains of 11q13.1-13.3 and losses of 18q12.1-23 in SCC. Furthermore, the expression levels of the sets of genes at both these loci in SCC were measured using microarray analysis. By combining the array-CGH with the microarray data, 10 genes at 11q13.1-13.3 and 6 genes at 18q12.1-23 whose expression correlated with chromosomal alterations were identified. To verify the expression levels of the identified genes, we used expression analysis data derived from our earlier study of clinical specimens. In clinical samples, six genes (GAL, GSTPI, MRPLII, MRPL2I, SF3B2, and YIFIA) at 11q13.1-13.3 and one gene (GALRI) at 18q23 showed a significant difference between normal and tumor samples. GAL, coding for the neuropeptide galanin, and GALR1, a galanin receptor, were identified as candidate genes of oncogenesis in SCC. The expression levels of GAL, GALR1, GALR2, and GALR3 were confirmed by real-time PCR. The expression ratio between GAL and GALR1 showed a significant negative correlation. GALR1 is a G-protein-coupled receptor that activates GTP-binding proteins to trigger signaling cascades such as the mitogen-activated protein kinase pathway, and is a well-established mitogenic pathway. This further supports the hypothesis that the genes involved in the GAL signaling cascade are candidates for regulation of oncogenesis in SCC. © 2008 Wiley-Liss, Inc.

Distant metastasis is a major factor associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), but little is known of its molecular mechanisms. New markers that predict clinical outcome, in particular the ability of primary tumors to develop metastatic tumors, are urgently needed. Based on a genome-wide gene expression analysis using clinical specimens of HNSCC, we narrowed our focus to the analysis of the neurotensin (NTS) and neurotensin receptor 1 (NTSR1) oncogenic signal pathways. Kaplan-Meier curves and log rank tests revealed that high mRNA expression levels of NTS and NTSR1 had a significant adverse effect on metastasis-free survival rate, suggesting a contribution of this pathway in HNSCC cancer progression. In HNSCC cells, which expressed NTSRl, a NTS agonist promoted cellular invasion, migration and induction of several mRNAs, such as interleukin 8 and matrix metalloproteinase 1 transcripts. In addition, knock down of NTSR1 expression with small interfering RNAs resulted in reduction of cellular invasion and migration in HNSCC cell lines. Our findings suggest a critical role for the NTS and NTSR1 oncogenic pathways in invasion and migration of HNSCC cells during the metastatic process. Our study raises the possibility that NTS and NTSR1 could be a useful predictive marker of poor prognosis in patients with HNSCC and a molecular therapeutic target in antimetastatic strategies for HNSCCs. © 2008 Wiley-Liss, Inc.

Prostate-specific antigen (PSA) screening has led to a remarkable increase in prostate cancer cases undergoing operative therapy. Over half of patients with locally advanced cancer (>= pT3) develop rising PSA levels (biochemical failure) within 10 years. It is very difficult to predict which patients will progress rapidly to advanced disease following biochemical failure (BF). Therefore, a more useful prognostic factor is needed to suggest the most appropriate therapies for each patient. To determine chromosomal aberrations, we examined 30 patients with stage pT2 or pT3 primary prostate adenocarcinomas and no metastases (pN0M0) by comparative genomic hybridization (CGH). Laser capture microdissection (LCM) was used to gather cancer cells from frozen prostate specimens. Common chromosomal alterations included losses on 2q23-24, 4q26-28, 6q14-22, 8p12-22 and 13q21-31, as well as gains on 1p32-36, 6p21 and 17q21-22. Losses at 8p12-22 and 13q21-31 were observed more frequently in pT3 than pT2 tumors (P<0.05 and P<0.01, respectively). Losses at 8p12-22 were more frequent in tumors with BF (P<0.05), and those at 13q12-21 were more frequent in tumors with Gleason score (GS) 7 or more than lower GS (P<0.05). These findings suggest that losses of 8p12-22 and 13q21-31 are important determinants of prostate cancer progression.

To determine the clinical significance of gene promoter methylation in esophageal squamous cell carcinoma (ESCC), we examined the promoter methylation status of genes showing elevated expression, as determined by DNA microarray-based transcriptomic analysis, in three ESCC cell lines (TE-1, TE-2, TE-10) after 5-aza-2'-deoxycytidine (DAC) treatment. We observed a high degree of DNA methylation within the promoter regions of three genes, namely cathepsin L2 (CTSL2), normal mucosa of esophagus specific 1 (NMES1), and fatty acid binding protein 5 (FABP5). Overexpression of NMES1 in ESCC cell lines increased cell motility. Downregulation of NMES1 might play an important role in the cell motility of ESCC or be a potent marker of malignancy.

Purpose: Aberrant DNA hypermethylation has been reported in renal cell carcinoma. We performed microarray analysis in the renal cancer cell line ACHN treated with the demethylating agent 5-aza-2'-deoxycytidine and investigated the UCHL1 gene involved in the regulation of cellular ubiquitin levels. Materials and Methods: We subjected 131 renal cell carcinoma and 61 corresponding normal kidney tissue samples to real-time reverse transcriptase-polymerase chain reaction, quantitative methylation specific polymerase chain reaction and immunohistochemistry. We also established a stable UCHL1 transfectant to evaluate cell growth. Results: We identified 10 genes that were up-regulated more than 2.5-fold in 5-aza-2'-deoxycytidine treated vs untreated ACHN cells. UCHL1 expression was increased 3.41-fold by 5-aza-2'-deoxycytidine treatment. In clinical samples the UCHL1 methylation index was significantly higher in renal cell carcinoma than in normal kidney tissue (p = 0.011). Conversely UCHL1 mRNA expression was significantly lower in renal cell carcinoma than in normal kidney tissue (p < 0.0001). There was a negative correlation between mRNA expression and the UCHL1 methylation index (p = 0.017). The immunostaining score for UCHL1 was significantly higher in normal kidney tissue than in renal cell carcinoma (p < 0.0001). Kaplan-Meier analysis showed that a positive UCHL1 methylation index had a significant adverse effect on prognosis (p = 0.048). Significant growth inhibition in UCHL1 transfectant compared to that in WT ACHN (p < 0.0001) suggests that UCHL1 functions as a potential tumor suppressor gene in human renal cell carcinoma. Conclusions: To our knowledge we report the first study demonstrating that the mechanism of UCHL1 down-regulation in renal cell carcinoma is through CpG hypermethylation of the promoter region and methylation of the UCHL1 gene is associated with a poor prognosis in patients with renal cell carcinoma.

Purpose The lack of treatment options other than surgical resection results in unfavourable prognosis of advanced gallbladder carcinoma. The aim of this study was to identify cancer-specific cellular targets that would form the basis for some therapeutic approaches for this disease. Methods Twelve advanced gallbladder carcinoma tissue samples and three samples of normal gallbladder epithelium were screened to identify differentially expressed genes by DNA microarray analysis. The results obtained were validated in an independent sample set by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Among the genes picked-up, one molecule, topoisomerase II alpha (TOPO II alpha), was further assessed immunohistochemically as a potential chemotherapeutic target, and the growth inhibitory effects of etoposide, doxorubicin and idarubicin, representative TOPO II alpha inhibitors, on two different gallbladder carcinoma cell lines were compared with that of gemcitabine and 5-fulorouracil. Results Five upregulated genes were identified: four cell cycle-related genes (TOPO II alpha, cyclin B2, CDC28 protein kinase regulatory subunit 2, ubiquitin-conjugating enzyme E2C) and a metabolism-related gene (gamma-glutamyl hydrolase). Immunohistochemically, TOPO II alpha was expressed in gallbladder cancer cells, and 16 of 35 cases (46%) had strong TOPO II alpha expression defined as having a labeling index of > 50%. In in vitro growth inhibition assay, etoposide, as well as doxorubicin and idarubicin, was the most effective for OCUG-1 cells that had strong TOPO II alpha expression, while gemicitabine was the most effective for NOZ cells with weak TOPO II alpha expression. Etoposide induced apoptosis of OCUG-1 cells. Conclusions TOPO II alpha might be an effective chemotherapeutic target in advanced gallbladder carcinoma, especially when it is expressed strongly.

Although the WT1 gene was originally isolated as a tumor suppressor gene from Wilms' tumor, oncogenic roles for WT1 have been reported in several tumors. Here, we present new findings of high levels of WT1 expression associated with the suppression of lymph node metastasis in patients with human lung squamous cell carcinoma (SCC). We investigated the effect of down-regulated WT1 gene expression on the invasive phenotype of the SCC cell line RERF-LC-AI. Invasive ability was enhanced in WT1-specific siRNA-transfected cells, and a WT1 target gene p21(waf1/Cip1) was isolated by comprehensive gene expression analysis. As several isoforms are produced from the WT1 gene, we isolated eight major WT1 isoforms from a cDNA library and cloned each variant into an expression vector. Luciferase reporter assays revealed that P21(waf1/cip1) expression was enhanced only by the WT1 cDNA variants that included a three-amino acid deletion (-KTS). Our results suggested that the -KTS-containing variants of WT1 are directly involved in the regulation of P21(waf1/Cip1) expression and the subsequent suppression of lymph node metastasis in human lung squamous cell carcinoma.

DNA amplifications activate oncogenes and are hallmarks of nearly all advanced cancers including head and neck squamous cell carcinoma (HNSCC). Some oncogenes show both DNA copy number gain and mRNA overexpression. Chromosomal comparative genomic hybridization and oligonucleotide microarrays were used to examine 8 HNSCC cell lines and a plot of gene expression levels relative to their position on the chromosome was produced. Three highly up-regulated genes, NT5C3, ANLN and INHBA , were identified on chromosome 7p14. These genes were subjected to quantitative real-time RT-PCR on cDNA and genomic DNA derived from 8 HNSCC cell lines. ANLN and INHBA showed a strong positive correlation between mRNA expression and genomic DNA levels and a similar relationship was shown for the known oncogene, EGFR, at 7p11.2. In clinical samples, ANLN and INHBA showed a significantly higher expression in tumors than in normal tissues. Patients with high expression levels of INHBA had a shorter disease-free survival rate. Therefore, INHBA may be a promising prognostic marker of HNSCC.

Using proteomic selection, functional verification, and clinical validation, we identified specific down-regulation of Lin-7C/ VELI3/MALS-3 (Lin-7C), which marks oral squamous cell carcinoma (OSCC) metastasis. Despite a rarity of sequence variations in the Lin-7C gene in both primary OSCC and OSCC-derived cells, a high prevalence of hypermethylation was detected in the CpG island region that strongly correlated with its down-regulation. Inducible Lin-7C mRNA by experimental demethylation was found in all OSCC cells tested. Overexpression of the Lin-7C gene in an OSCC cell clone does not contribute to underproliferation but results in a noninvasive phenotype with elevated β-catenin expression. Experimental metastases in multiple organs of immunodeficient mice were inhibited in cells expressing Lin-7C. Finally, the Lin-7C expression status in primary tumors afforded significantly (P &lt 0.001) high accuracy for predicting lymph node metastasis. These results establish Lin-7C as a novel target of early detection, prevention, and therapy for OSCC metastasis. ©2007 American Association for Cancer Research.

Arsenic trioxide (As2O3) has been used with success in the treatment of acute promyelocytic leukemia. However, resistance to arsenite agents reduces their efficacy. We have isolated arsenite-resistant human epidermoid carcinoma KB cells, termed KAS. KAS cells were resistant to sodium arsenite (22-fold) and showed a reduced accumulation of arsenite as a result of an active efflux mechanism. Further analysis indicated that resistance of KAS cells extended to other drugs including cisplatin (17-fold), antimony potassium tartrate (11-fold) and doxorubicin (27-fold). Although increased expression of multidrug resistance protein I (MRP1) in KAS cells was confirmed by quantitative RTPCR and immunoblot analysis, specific inhibitors of MRP1 did not completely eliminate arsenite resistance. The level of glutathione (GSH) in KAS cells was 3-fold higher than that in KB-3-1 cells, and the inhibition of GSH synthesis by buthionine sulfoximine (BSO) considerably increased the cytotoxic effect of arsenite on KAS cells. A pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl ethyl 5(trans-4, 6-dimethyl- 1,3,2-dioxaphosphorinan-2-yl)-2, 6dimethyl-4-(3-nitrophenyl)-3-pyridine-carboxylate P oxide (PAK-104P), partially reversed the arsenite resistance and increased the arsenite accumulation in KAS cells. We suggest that the increased level of GSH is involved in arsenite resistance and an as yet unidentified arsenite transporter is expressed in the arsenite-resistant KAS cells.

Background: Substantial evidence has disclosed that some cytotoxic agents have complex activities in influencing signal transduction pathways in cells. Materials and Methods: cDNA microarray analysis was performed after exposing a human squamous cell carcinoma cell line, RERF-LC-AI, to low-dose cisplatin for 5 days. Up-regulated gene expressions were suppressed by small interfering RNA to investigate phenotypic alteration of the cells. Results: Among 30,000 genes screened, 42 genes showed increases or decreases in expression of more than 2-fold with cisplatin treatment. They included genes with functions involved in apoptosis, cell cycle regulation and DNA metabolism/repair. Suppression of the 5 most significantly altered genes by small interfering RNA resulted in partly reduced apoptosis without altering cytotoxicity of cisplatin. Conclusion: Besides direct cytotoxic effects on cells, cisplatin may have indirect effects involving drug resistance, and synergistic effects with other agents.

Microarray and comparative genomic hybridization (CGH) studies have provided a wide range of information about esophageal cancer, but the correlations between gene expression and copy number alteration are largely unknown. To identify putative amplification target genes in esophageal cancer, a survey of parallel DNA copy number and gene expression in 10 esophageal squamous cell carcinoma (ESCC) cell lines was performed using classical CGH and oligonucleotide microarrays. The gene expression and copy number data were subsequently integrated using signal-to-noise ratio analysis. The results revealed a set of 97 genes with elevated expression levels that were attributable to increased copy number. The set included genes previously reported as overexpressed in cancer as well as several novel genes associated with copy number elevation. These genes are involved in essential cellular processes (e.g., regulation of transcription, signal transduction, cell proliferation, the cell cycle and cell differentiation) that can also have an impact on cancer development. Thus, the integration of DNA and RNA profiles provides a highly productive entry point for the discovery of genes involved in the development and progression of esophageal cancer.

Purpose: SKP2 and CKS1 promote aggressive tumor behavior via the regulation of p27 degradation. Our previous DNA microarray analysis of human urothelial carcinoma and normal urothelial epithelium showed that in urothelial carcinoma the 2 most highly up-regulated genes among SKP2-p27 interaction related genes are SKP2 (4.7-fold) and CKS1 (2.2-fold). We hypothesized that SKP2 and CKS1 gene expression is associated with urothelial carcinoma invasiveness and prognosis. Materials and Methods: A total of 84 urothelial carcinoma specimens from patients with bladder (71) and upper urinary tract (13) cancer were examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemical. study. Results: Real-time reverse transcriptase-polymerase chain reaction showed that the average mRNA expression level of SKP2 and CKS1 significantly correlated with tumor stage, that is superficial vs invasive urothelial carcinoma (SKP2 and CKS1, p <0.001 and 0.006) and grade (p <0.001 and 0.009, respectively). Of the superficial urothelial carcinomas examined the SYP2 and CKS1 expression level was significantly higher in pT1 than in pTa samples (p = 0.005 and 0.017, respectively). Immunohistochemical expression patterns of SKP2 and CKS1 also significantly correlated with tumor stage (p <0.001 and 0.048) and grade (p = 0.003 and 0.025, respectively). In contrast, p27 expression inversely correlated with tumor stage and grade (p <0.001 and 0.011, respectively). Logistic regression analysis revealed that while SKP2 mRNA expression was a significant dependent predictor of p27 expression (p = 0.021), there was no correlation between CKS1 mRNA expression and p27 (p = 0.748). Kaplan-Meier curves and log rank tests revealed that the high mRNA expression levels of SKP2 and CKS1 had a significant adverse effect on prognosis (p = 0.043 and 0.003, respectively). Conclusions: Our results suggest that SKP2 has a major role in the regulation of p27 degradation and CKS1 has a supporting role for SKP2 function in human urothelial carcinoma.

Ku80 is an important component of DNA double-strand break repair, and Ku80 deficiency leads to extreme sensitivity to ionizing radiation. We studied whether radiation therapy combined with Ku80 silencing by small interfering RNA enhances radiation sensitivity in vitro and in vivo. Seven human cancer cell lines were transfected with Ku80 siRNA included in hemagglutinating virus of Japan envelope vector. H1299 cells were implanted into male BALB/C nu/nu nude mice treated with Ku80 siRNA and irradiation. The survival rate of cell lines transfected with Ku80 siRNA decreased by 10% to 26% with 2-Gy irradiation compared with untransfected cell lines. The gamma-H2AX phosphorylation-positive rates of Ku80 siRNA combined treatment 0.5 h after irradiation in A549 cells and 6 h in H1299 cells were significantly higher (77.6%, p=0.033 and 76.7%, p=0.026, respectively), compared with the groups not treated with siRNA. H1299 xenograft tumors treated with combined therapy decreased in volume and re-grew slowly compared with radiation alone. Our results indicate that combined therapy consisting of Ku80 siRNA and irradiation contributes to inhibition of tumor growth and may be a novel strategy for cancer treatment.

Pathogens of the Aspergillus species are frequently seen in deep-seated mycoses. We previously demonstrated that the culture filtrate of Aspergillus fumigatus (CF) has immunosuppressive effects on polymorphonuclear leukocytes (PMNs), which act as the main phagocytes to hyphae of Aspergillus fumigatus (A. fumigatus). But little is known about the gene expression profiles involved in it. Therefore we investigated the changes in gene expression in human PMNs treated with CF or gliotoxin at two time points, using microarray analysis. CF and gliotoxin changed the expression of 548 and 381 genes, respectively. Only 51 genes showed the same expression patterns with the two stimulants, and CF-induced changes in gene expression occurred comparatively earlier than those induced by gliotoxin. Among 31 genes encoding apoptosis, which were up- or down-regulated in this assay, only 3 genes were similarly changed by both kinds of stimulation. Apoptosis was detected and quantified using two apoptosis assays. CF and gliotoxin changed the expessions of only 3 out of 19 regulated genes related to inflammatory mediators and receptors similarly. The up-regulation of the gene encoding annexin 1 (ANXA1), which is known to be involved in extravasation and apoptosis of neutrophils, may play a role in the immunosuppressive effect of A. fumigatus. The difference in expression changes between CF and gliotoxin is presumed to be caused by the interaction among the components of CF and therefore the interaction is an area of interest for further investigation.

Background/Aims: Only limited patients with hepatoma. benefit from chemotherapy without a clear explanation. We aimed to identify genes associated with chemosensitivity using transcriptional profiles. Methodology: In 8 hepatoma cells (HLE, HLF, Huh7, Hep3B, PLC/PRF/5, SK-Hep1, Huh6, and HepG2) transcriptional profiles were obtained using cDNA microarray including 2,300 genes. Chemosensitivities to 8 anticancer drugs (nimustine, mitomycin C, cisplatin, carboplatin, doxorubicin, epirubicin, mitoxantrone, and 5-fluorouracil) were measured by obtaining 50% growth inhibitory concentrations (GI50) using MTT assay. Genes having drug-specific association with chemosensitivity were selected. Results: Up-regulation of topoisomerase II beta was associated with chemo-resistance, the target of doxorubicin. Platinum-specific resistance was associated with superoxide dismutase 2 expression. Antigen peptide transporter I expression correlated with nimustine and mitoxantrone-specific susceptibility. These results were verified by semi-quantitative RT-PCR. Drug inactivators reported in non-liver cancers such as multidrug transporters and drug metabolizers showed less diversity of chemosensitivity in hepatoma cells. Conclusions: To evaluate these gene expressions may be useful to select anticancer drugs, and possibly to consider new therapeutic target to modify drug action.

Objective. Aberrant methylation of promoter CpG islands is associated with the loss of expression of tumor suppressor genes in human cancers. The purpose of this study was to examine methylation-silenced genes in colorectal cancer ( CRC) cell lines. Material and methods. Using an oligonucleotide array, we undertook a genome-wide search for genes upregulated following treatment with a demethylating agent ( 5-aza-2'-deoxycytidine) in two CRC cell lines, DLD-1 and HT-29. Promoter methylation status was determined in 12 CRC cell lines and 11 CRC tissues by methylation-specific polymerase chain reaction ( MSP). Results. After treatment, 350 genes were up-regulated 1.5-fold or more. Six genes ( PAGE-5, VCX, MAEL, GAGED2, UCHL1, and GAGE7), which contained putative 5' CpG islands in their promoter regions, were confirmed to be silenced in CRC cell lines. UCHL1 ( also known as PGP9.5) is involved in regulation of cellular ubiquitin levels, and its promoter methylation was detected in 10 out of 12 CRC cell lines. The level of methylation of UCHL1 was significantly higher in tumors than in corresponding normal mucosae ( p = 0.005). Conclusions. Chemical genomic screening led to the identification of a specific promoter subject to hypermethylation in CRC. These results suggest that aberrant promoter methylation is the primary mechanism of transcriptional silencing of the UCHL1 gene and that methylation of the UCHL1 gene promoter increases during the development and progression of CRC.

Treatment protocols for malignant tumors in the oral cavity differ greatly based on the presence of cervical lymph node metastasis. We applied gene expression profiling to the pathological lymph node status and used a training-test approach to evaluate the reliability of cDNA microarray-based classifications of 15 matched resected primary oral squamous cell carcinomas (OSCCs) and corresponding normal oral tissues. The clustering of all the microarray data was separated into two groups based on metastatic node positivity and node negativity. Furthermore, a 20-gene signature was identified that differentiated the testing set (n=8) with high classification accuracy (88%). Our findings support the hypothesis that the lymph node metastasis status can be predicted using the gene expression patterns of the primary OSCC, and may be a powerful tool in identifying patients at high risk of lymph node metastasis.

Several mutations of the tyrosine kinase domain of insulin receptor (IR) have been clinically reported to lead insulin resistance and insulin hypersecretion in humans. However, it has not been completely clarified how insulin resistance and pancreatic β-cell function affect each other under the expression of mutant IR. We investigated the response of pancreatic β-cells in mice carrying a mutation (P1195L) in the tyrosine kinase domain of IR β-subunit. Homozygous (IrP1195L/ P1195L) mice showed severe ketoacidosis and died within 2 days after birth, and heterozygous (IrP1195L/wt) mice showed normal levels of plasma glucose, but high levels of plasma insulin in the fasted state and after glucose loading, and a reduced response of plasma glucose lowering effect to exogenously administered insulin compared with wild type (Irwt/wt) mice. There were no differences in the insulin receptor substrate (IRS)-2 expression and its phosphorylation levels in the liver between IrP1195L/wt and Irwt/wt mice, both before and after insulin injection. This result may indicate that IRS-2 signaling is not changed in IrP1195L/wt mice. The β-cell mass increased due to the increased numbers of β-cells in IrP1195L/wt mice. More proliferative β-cells were observed in IrP1195L/wt mice, but the number of apoptotic β-cells was almost the same as that in Irwt/wt mice, even after streptozotocin treatment. These data suggest that, in IrP1195L/wt mice, normal levels of plasma glucose were maintained due to high levels of plasma insulin resulting from increased numbers of β-cells, which in turn was due to increased β-cell proliferation rather than decreased β-cell apoptosis. © 2006 Society for Endocrinology.

Large-scale gene expression profiling is an effective strategy for understanding the progression of bladder cancer (BC). The aim of this study was to identify genes that are expressed differently in the course of BC progression and to establish new biomarkers for BC. Specimens from 21 patients with pathologically confirmed superficial (n=10) or invasive (n=11) BC and 4 normal bladder samples were studied; samples from 14 of the 21 BC samples were subjected to microarray analysis. The validity of the microarray results was verified by real-time RT-PCR. Of the 136 up-regulated genes we detected, 21 were present in all 14 BCs examined (100%), 44 in 13 (92.9%), and the other 71 in 12 BCs (85.7%). Of 69 down-regulated genes, 25 were found in all 14 BCs (100%), 22 in 13 (92.9%), and the other 22 in 12 BCs (85.7%). Functional annotation revealed that of the up-regulated genes, 36% were involved in metabolism and 14% in transcription and processing; 25% of the down-regulated genes were linked to cell adhesion/surface and 21% to cytoskeleton/cell membrane. Real-time RT-PCR confirmed the microarray results obtained for the 6 most highly up- and the 2 most highly down-regulated genes. Among the 6 most highly up-regulated genes, CKS2 was the only gene with a significantly greater level of up-regulation in invasive than in superficial BC (p=0.04). To confirm this result, we subjected all 21 BC samples to real-time PCR assay for CKS2. We found a considerable difference between superficial and invasive BC (p=0.001). Interestingly, there was a considerable difference between the normal bladder and invasive BC (p=0.001) and less difference between the normal bladder and superficial BC (p=0.005). We identified several genes as promising candidates for diagnostic biomarkers of human BC and the CKS2 gene not only as a potential biomarker for diagnosing, but also for staging human BC. This is the first report demonstrating that CKS2 expression is strongly correlated with the progression of human BC.

PURPOSE. Choroidal malignant melanomas (CMMs) are the most common primary intraocular tumors in adult humans. Although radiotherapy is commonly used to treat the melanomas, the therapeutic effects are unpredictable. The purpose of this study was to search for a gene(s) that can predict the success of radiotherapy for CMMs. METHODS. The cell lines 92-1, OCM-1, and OMM-1 were established from patients with CMM, and radiation sensitivity was determined using the colony-formation assay. RNA was extracted from nonirradiated cells, and gene expression analysis was performed using a microarray containing 10,800 genes. The up- or downregulated genes were verified by real-time PCR using other cancerous cell lines in which radiation sensitivity had been documented. RESULTS. Analysis of radiation survival curves showed that cell line 92-1 was radiation sensitive and OCM-1 and OMM-1 lines were radiation resistant. The results of microarray analyses showed that 34 genes were differentially expressed in the OCM-1 and OMM-1 cell lines compared with the 92-1 cell line. The validity of the expression level of 13 of the 34 genes that were identified by microarray was confirmed by PCR. From the analysis of the different radio-sensitivity cancer cell lines, the Arpc1b gene was selected as a prediction marker gene for sensitivity of CMM to radiotherapy. CONCLUSIONS. Gene expression analysis of CMM cell lines can be used to search for radiation sensitivity prediction markers. Comprehensive gene expression profiles of radiation-sensitive and/or resistant cell lines may provide new insights into the mechanisms of resistance or sensitivity to radiation therapy. Copyright © Association for Research in Vision and Ophthalmology.

This study was designed to disclose detailed genetic mechanisms in salivary gland tumors (SGTs) for development of novel independent marker. We constructed an in-house cDNA microarray carrying 2,201 cDNA clones derived from SGT and oral squamous cell carcinoma cDNA libraries. Four cell lines that originated from the SGT-derived cell lines were analyzed using this microarray system. The genes identified by our microarray system were further analyzed at the mRNA or protein expression level in other types of human cancer cell lines and clinical samples (ten normal salivary glands [NSGs], eleven pleomorphic adenomas, ten adenoid cystic carcinomas and three adenocarcinomas). Two up-regulated genes and six down-regulated genes were identified in common when compared with the control RNA. Of the up-regulated genes, WISP-2, which plays an important role in breast carcinogenesis, was selected for further analyses. We found a higher expression of the WISP-2 gene in the SGT-derived cell lines compared with other types of human cancer cell lines. Furthermore, WISP-2 mRNA and protein expression levels in NSGs were significantly higher than those in SGTs. These results suggest that WISP-2 could be a reliable independent marker and that down-regulation or loss of the WISP-2 gene may be associated with the development of SGTs.

Objective. Fulminant hepatic failure (FHF) is a clinical syndrome of sudden and severe liver dysfunction accompanied by encephalopathy in a previously healthy person. In FHF, hepatocytes are severely damaged and ordinary liver regeneration is impaired. We demonstrated that the expression of osteopontin (OPN), a multifunctional cytokine, was up-regulated in mouse oval cell (a stem-cell progenitor) induction models. Material and methods. Based on this finding, serum OPN levels were examined in 43 patients with FHF and in 45 patients with acute self-limited hepatitis (AH). To determine the cellular source of OPN, the expression of OPN was studied in a liver specimen derived from an FHF patient. Results. The mean OPN level of patients with FHF was 2.80±0.48 ng/ml (log 10, ±SD), which was significantly higher than that of the patients with AH (2.42±0.39 ng/ml) (p = 0.003, unpaired t-test). Patients with elevated serum OPN levels had a significantly poorer prognosis than patients whose serum OPN levels were not elevated. In the FHF patient's liver, OPN protein was expressed not only in inflammatory cells but also in regenerating hepatocytes and bile ductular structures. Conclusions. Our current study indicates that serum OPN levels increased in patients with FHF and that OPN might play an important role in liver regeneration due to activation of hepatic stem cells. © 2006 Taylor &amp Francis.

Purpose: The histone deacetylase inhibitor FK228 shows strong activity as a potent antitumor drug but its precise mechanism is still obscure. The purpose of this study is to reveal the effect of FK228 on gene expression in the cell and to determine the mechanism of the antitumor activity of FK228 for further clinical applications. Experimental Design and Results: Microarray analysis was applied to verify the gene expression profiles of 4,608 genes after FK228 treatment using human esophageal squamous cell cancer cell lines TTn and TE2. Among them, peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family of antioxidant enzymes having cell growth suppression activity, as well as p21 WAF1, were significantly activated by FK288. In addition, FK228 strongly inhibited the cell growth of TTn and TE2 by the induction of apoptosis. Further, chromatin immunoprecipitation analysis revealed that FK228 induced the accumulation of acetylated histones H3 and H4 in Prdx1 promoter, including the Sp1-binding site. In mouse xenograft models of TTn and TE2 cells, FK228 injection resulted in significant tumor regression as well as activated Prdx1 expression in tumor tissues. Prdx1 suppression by RNA interference hindered the antitumor effect of FK228. Conclusion: Our results indicate that the antitumor effect of FK228 in esophageal cancer cells is shown at least in part through Prdx1 activation by modulating acetylation of histones in the promoter, resulting in tumor growth inhibition with apoptosis induction. © 2005 American Association for Cancer Research.

AIM: To elucidate the sequential gene expression profile in AGS cells co-cultured with wild-type Helicobacter pylori (H pylori) as a model of H pylori-infected gastric epithelium, and to further examine the contribution of cag-pathogenicity islands (cagPAI)-coding type IV secretion system and the two pathways, nuclear factor kappa B (NF-kappa B) and extracellular signal-regulated kinases (ERK) on wild-type H pylori-induced gene expression. METHODS: Gene expression profiles induced by H pylori were evaluated in AGS gastric epithelial cells using cDNA microarray, which were present in the 4 600 independent clones picked up from the human gastric tissue. We also analyzed the contribution of NF-kappa B and ERK signaling on H pylori-induced gene expression by using inhibitors of specific signal pathways. The isogenic mutant with disrupted cagE (Delta cagE) was used to elucidate the role of cagPAI-encoding type IV secretion system in the gene expression profile. RESULTS: According to the expression profile, the genes were classified into four clusters. Among them, the clusters characterized by continuous upregulation were most conspicuous, and it contained many signal transducer activity-associated genes. The role of cagPAI on cultured cells was also investigated using isogenic mutant cagE, which carries non-functional cagPAI. Then the upregulation of more than 80% of the induced genes (476/566) was found to depend on cagPAI. Signal transducer pathway through NF-kappa B or ERK are the major pathways which are known to be activated by cagPAI-positive H pylori. The role of these pathways in the whole signal activation H pylori was analyzed. The specific inhibitors against NF-kappa B or ERK pathway blocked the activation of gene expression in 65% (367/566) or 76% (429/566) of the genes whose activation appealed to depend on cagPAI. CONCLUSION: These results suggest that more than half of the genes induced by cagPAI-positive H pylori depend on NF-kappa B and ERK signaling activation, and these pathways may play a role in the gene expression induced by host-bacterial interaction which may associate with H pylori-related gastro-duodenal diseases. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved.

Esophageal and oral carcinomas are relatively resistant to adenovirus serotype 5 (Ad5)-mediated gene transfer, primarily because expression of the cellular receptors for Ad5, the coxsackievirus and adenovirus receptor, is often dowunregulated in these types of tumor. The types of Ad in which the receptor expression is not suppressed in tumors are therefore better vectors for gene transfer into tumors. CD46, a cellular receptor for Ad subtype B2, such as Ad11 and Ad35, is well expressed in a number of esophageal and oral tumor cells. Since the infectivity of Ad to target cells is mainly influenced by the interaction between their fibers and the cellular receptors, we examined the infectivity of chimeric Ad5, whose fiber structure was substituted with that of type 11 or 35 (Ad5/11 or Ad5/35), to 6 human oral and 11 esophagus carcinoma cells. We found that the chimeric Ad, in particular Ad5/35, infected more effectively than Ad5 in all the tumors tested. However, the efficacy of Ad5/35- and Ad5/11-mediated transduction was not correlated with the expression level of CD46 or CD80/86, a cellular receptor of the Ad subtype B1, in the target cells. These data suggest that the Ad subtype B2 are suitable vectors of gene transfer for human squamous cell carcinomas of the upper gastrointestinal tract, and that the infectivity of the Ad subtype B2 can possibly be regulated by other receptors besides CD46.

NeuroD-related factor (NDRF)/NeuroD2 is a basic helix-loop-helix (bHLH) protein that plays important roles in neuronal development. To elucidate the NDRF transcription network, we used mouse cDNA microarray analysis combined with a tetracycline-regulatable expression system in P19 embryonal carcinoma cells. Five genes were identified to be up-regulated in the presence of NDRF protein. RNA hybridization analysis confirmed that brain-lipid-binding protein (BLBP) and inhibitor of differentiation 1 (Id1) genes were among the five genes that were rapidly and significantly up-regulated after induction of NDRF. When a dominant negative form of NDRF protein was expressed during retinoic acid-induced neuronal differentiation of P19 cells, the BLBP gene, but not the Id1 gene, was potently repressed. Immunohistochemical analysis revealed that both NDRF and Id1 immunoreactivities were observed in some granule cells of the cerebellum in the postnatal period. These results suggest that NDRF or its related bHLH proteins may act upstream of these genes in a subset of developing neurons. © 2005 Elsevier Inc. All rights reserved.

Sjogren's syndrome is a chronic autoimmune disease characterized by focal lymphocytic infiltration of lacrimal and salivary glands, but the precise mechanism of this syndrome is unclear. To clarify the pathogenesis of Sjogren's syndrome, the related genes must be identified. In the present study, we investigate the increased expression of genes and molecules related to Sjogren's syndrome and present our findings of cDNA microarray analysis in the mouse model. Furthermore, we present the results of immunohistochemical analysis of salivary glands in the mouse model and patients with Sjogren's syndrome. This approach might open a new discussion of the existence of principal pathogenic molecules in Sjogren's syndrome. (c) 2005 Elsevier B.V. All rights reserved.

This study was designed to identify specific gene expression changes in tongue squamous cell carcinomas (TSCCs) compared with normal tissues using in-house cDNA microarray that comprised of 2304 full-length cDNAs from a cDNA library prepared from normal oral tissues, primary oral cancers, and oral cancer cell lines. The genes identified by our microarray system were further analysed at the mRNA or protein expression level in a series of clinical samples by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis and imuunohositochemistry. The microarray analysis identified a total of 16 genes that were significantly upregulated in common among four TSCC specimens. Consistent with the results of the microarray, increased mRNA levels of selected genes with known molecular functions were found in the four TSCCs. Among genes identified, Rab1a, a member of the Ras oncogene family, was further analysed for its protein expression in 54 TSCCs and 13 premalignant lesions. We found a high prevalence of Rab1A-overexpression not only in TSCCs (98%) but also in premalignant lesions (93%). Thus, our results suggest that rapid characterisation of the target gene(s) for TSCCs can be accomplished using our in-house cDNA microarray analysis combined with the qRT-PCR and immunohistochemistry, and that the Rab1A is a potential biomarker of tongue carcinogenesis. © 2005 Cancer Research UK.